This invention relates to an endotoxin adsorbent and a method of removing endotoxin by using the same, and in particular to an endotoxin adsorbent whose exclusion limit molecular weight has been controlled and a method of removing endotoxin by using the same.
The term endotoxin is a generic name of toxic substances present in microbial components in microorganisms and released after the microorganisms perish. A structural component of endotoxin is a lipopolysaccharide produced in microorganisms living in a production process and mixed in pharmaceutical preparations after the microorganisms perish. Known methods of removing endotoxin include a method of using an adsorbent such as charcoal or an ion exchanger, a filtration method of using a membrane or a membrane filter, and a degradation method of using high temperature/high pressure treatment or an acid or alkali. Any methods have both advantages and disadvantages and are problematic for industrial use. For example, removal of endotoxin in production of pharmaceutical preparations cannot be conducted under severe conditions because of the necessity for maintaining the stability of original pharmaceutical preparations, and even if endotoxin can be adsorbed successfully in laboratories, endotoxin cannot be satisfactorily adsorbed in the industrial scale or pharmaceutical preparations themselves are adsorbed because of a very small amount of occurring endotoxin, and thus the conventional methods are not necessarily satisfactory.
On the other hand, some adsorbents have been proposed in recent years in order to enable endotoxin adsorption which is also satisfactory in industry. Japanese Patent Publication No. 16843/1994 discloses an adsorbent made of polyamino acid containing a modifying group having an aliphatic group and/or an aryl group at the terminal of a side chain and/or main chain thereof, and Japanese Patent Laid-Open No. 127039/1989 discloses an adsorbent comprising an imidazole derivative bonded to spherical particles of polyamino acid as carriers.
Further, Bulletin of the Chemical Society of Japan, No. 8, pp. 726-730 (1994) has reported the results of a study on the endotoxin-adsorbing ability of an adsorbent comprising polyethylene-imine fixed by using a crosslinking agent onto cellulose fibers.
In addition, J. LIQ. CHROM. and REL. TECHNOL., 23(12), 1887-1902 (2000) has reported the results of a study on the adsorbability of an endotoxin adsorbent using N,N-dimethylaminopropyl acrylamide, N-propylene acrylamide or N,Nxe2x80x2-butylene-bis-methacrylamide (BBMA) as a base material and divinyl benzene as a crosslinking agent.
However, these adsorbents are poor in selectivity for removing only endotoxin from solutions such as endotoxin-containing pharmaceutical preparations or blood containing proteins at high concentration, and there is the problem that acidic proteins which should not be adsorbed are adsorbed together with endotoxin.
In view of the problems in the prior art described above, the present inventors made extensive study. As a result, they found that an adsorbent with a controlled exclusion limit molecular weight comprising a specific basic substance bonded to a specific base material by means of a crosslinking agent, has a high ability to adsorb endotoxin selectively, and this adsorbent can be used to remove endotoxin highly selectively from solutions such as endotoxin-containing pharmaceutical preparations or blood containing proteins at high concentration, and on the basis of this finding, this invention have been completed.
That is, this invention encompasses:
(1) an endotoxin adsorbent with an exclusion limit molecular weight of 6000 or less as determined by polysaccharide standards, which comprises a basic substance bonded to a base material by means of a crosslinking agent;
(2) the endotoxin adsorbent according to the above-mentioned (1), wherein the base material is a polysaccharide selected from cellulose, agarose, chitosan, chitin, starch and mannan, a derivative of said polysaccharide, a polymer of a monomer selected from acrylamide, methacrylamide, acrylic acid, acrylate, methacrylic acid, methacrylate, vinyl acetate, vinyl chloride, styrene and an allyl compound, or a copolymer of two or more of these monomers;
(3) the endotoxin adsorbent according to the above-mentioned (1), wherein the base material is a polysaccharide selected from cellulose, agarose, chitosan, chitin, starch and mannan, a derivative of said polysaccharide, a polymer of a monomer selected from acrylic acid, acrylate, methacrylic acid, methacrylate, vinyl acetate, vinyl chloride, styrene and an allyl compound, or a copolymer of two or more of these monomers;
(4) the endotoxin adsorbent according to the above-mentioned (1), wherein the base material is cellulose or a derivative thereof;
(5) the endotoxin adsorbent according to any one of the above-mentioned (1) to (4), wherein the base material is porous cellulose or a derivative thereof having an exclusion limit molecular weight of 6000 or less as determined by saccharide standards;
(6) the endotoxin adsorbent according to any one of the above-mentioned (1) to (4), wherein the base material is porous cellulose or a derivative thereof having an exclusion limit molecular weight of 2000 or less as determined by saccharide standards;
(7) the endotoxin adsorbent according to the above-mentioned (1), wherein the basic substance is at least one member selected from xcex5-poly-L-lysine, xcex1-poly-L-lysine, poly-histidine, poly-arginine, poly-tryptophan, polyornithine, poly-2,4-diaminobutyric acid, poly-2,3-diaminopropionic acid, protamine, polyallylamine, an allylamine-diallylamine copolymer, polydiallylamine, polyvinylamine, polymyxin, streptomycin, amikacin, and kanamycin.
(8) the endotoxin adsorbent according to the above-mentioned (1), wherein the basic substance is xcex5-poly-L-lysine or xcex1-poly-L-lysine;
(9) the endotoxin adsorbent according to the above-mentioned (1), wherein the crosslinking agent is at least one crosslinking agent selected from hexamethylene diisocyanate, m-xylene diisocyanate, toluene-2,4-diisocyanate, diglycidyl ether, epichlorohydrin and halogenated epichlorohydrin; and
(10) a method of removing endotoxin, which comprises using an endotoxin adsorbent described in any one of the above-mentioned (1) to (9).